Method for diagnosing and treating schizophrenia

ABSTRACT

The gene encoding decidual protein induced by progesterone (DEPP), the gene encoding adrenomedullin and the gene encoding cold shock domain protein A (csdA), are upregulated in the anterior cingulate of schizophrenic patients as compared to normal patients. Methods of screening, diagnosing and treating schizophrenia based on these genes are provided. Transgenic nonhuman animals having increased copy number or increased expression levels of these genes are also provided. The transgenic nonhuman animals are used in methods of screening for potential therapeutic agents.

BACKGROUND

1. Field of the Invention

The present disclosure relates to genes correlated to schizophrenia andmethods of using genes for diagnosis and treatment of schizophrenia.

2. Description of Related Art

Schizophrenia is a severe psychiatric disorder usually characterized bywithdrawal from reality, illogical patterns of thinking, delusions andhallucinations, and accompanied in varying degrees by other emotional,behavioral, or intellectual disturbances. See Diagnostic and StatisticalManual of Mental Disorders, Fourth Edition, American PsychiatricAssociation, 273-315 (1994) (DSM-IV™). However, as stated therein, nosingle symptom is pathognomonic of schizophrenia; the diagnosis involvesrecognition of a constellation of signs and symptoms associated withimpaired occupational or social functioning. Id. Some detectablephysiological changes have been reported, e.g., neuropathological andimaging studies depicting anatomical alterations associated with thedisease. Arnold et al., Acta Neuropathol. (Berl) 92, 217-231 (1996);Harrison, Brain 122, 593-624 (1999). Certain cellular aberrations havebeen observed and biochemical and RNA analyses have demonstratedalterations in some neurotransmitter pathways and presynapticcomponents. Id.; Benes, Brain Res. Rev. 31, 251-269 (2000).

At beginning stages and even at more advanced stages, schizophrenia caninvolve subtle behavioral changes and subtle and/or undetectable changesat the cellular and/or molecular levels in nervous system structure andfunction. This lack of detectable neurological defect distinguishesschizophrenia from other well-defined neurological disorders in whichanatomical or biochemical pathologies are clearly manifest. Thus, thereis a need for non-subjective modalities for screening and diagnosis ofschizophrenia. Moreover, identification of the causative defects and theneuropathologies of schizophrenia are needed in order to enableclinicians to evaluate and prescribe appropriate courses of treatment tocure or ameliorate the symptoms of schizophrenia at early stages or whensymptoms are obscured. Indeed, there are few effective therapies for thedisease and its molecular basis is still not well understood.

Methods have been designed to survey alterations in mRNA expression inorder to search for genes disregulated in various diseases anddisorders. In organisms for which the complete genome is known, it ispossible to analyze the transcripts of all genes within the cell. Withother organisms, such as human, for which there is an increasingknowledge of the genome, it is possible to simultaneously monitor largenumbers of genes within a cell. DNA microarray analysis is a techniquethat permits the quantitative measurement of the transcriptionalexpression of several thousand genes simultaneously. This techniquepermits one to generate profiles of gene expression patterns in bothpatients suffering from schizophrenia and control individuals.Accordingly, determination of abnormal levels of gene expressionprovides a signpost for therapeutic intervention.

Techniques for modifying RNA levels and activities involve ribozymes,antisense species, and RNA aptamers and small molecule promotermodulators. Ribozymes are RNAs capable of catalyzing RNA cleavagereactions, and some can be designed to specifically cleave a particulartarget mRNA. Ribozyme methods include exposing a cell to, inducingexpression in a cell, etc. of such RNA ribozyme molecules. Activity of atarget RNA (preferably mRNA) species, specifically its rate oftranslation, can be inhibited by the application of antisense nucleicacids. “Antisense” nucleic acids are nucleic acids capable ofhybridizing to a sequence specific portion of the target RNA, e.g., itstranslation initiation region by virtue of some sequence that iscomplementary to a coding and/or non-coding region. The antisensenucleic acid can be oligonucleotides that are double-stranded orsingle-stranded, RNA or DNA or a modification or derivative thereof,which can be produced intracellularly by transcription of exogenous,introduced sequences in controllable quantities sufficient to perturbtranslation of the target RNA.

The above described techniques are emerging as an effective means forreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic andresearch applications for the modulation of genes that are disregulatedin schizophrenic patients.

SUMMARY

In one aspect, a method for screening for schizophrenia in a populationis provided which includes determining, in members of the population,the magnitude of expression of a gene selected from the group consistingof the gene encoding decidual protein induced by progesterone (DEPP),the gene encoding adrenomedullin and the gene encoding cold shock domainprotein A (csdA) In a sample and comparing the magnitude of expressionto a baseline magnitude of expression of the gene, wherein increasedgene expression indicates the presence of schizophrenia. The sample maybe taken from the brain, spinal cord, lymphatic fluid, blood, urine orfeces. In a preferred embodiment the sample is taken from the anteriorcingulated. In another preferred embodiment, the population is human. Inanother aspect, a method for diagnosing schizophrenia in a host isprovided which includes determining the magnitude of expression of agene selected from the group consisting of the gene encoding decidualprotein induced by progesterone (DEPP), the gene encoding adrenomedullinand the gene encoding cold shock domain protein A (csdA) in a sample andcomparing the magnitude of expression to a baseline magnitude ofexpression of the gene, wherein increased gene expression indicates thepresence of schizophrenia.

In another aspect, a method for treating schizophrenia in a host isprovided which includes lowering expression of a gene selected from thegroup consisting of the gene encoding decidual protein induced byprogesterone (DEPP), the gene encoding adrenomedullin and the geneencoding cold shock domain protein A (csdA) by administering to the hostan expression lowering amount of antisense oligonucleotide or anexpression lowering amount small-inhibitory RNA (siRNA). In a preferredembodiment of this aspect, the host is human.

In another aspect, a method for treating schizophrenia in a host isprovided which includes lowering expression of a gene selected from thegroup consisting of the gene encoding decidual protein induced byprogesterone (DEPP), the gene encoding adrenomedullin and the geneencoding cold shock domain protein A (csdA) by administering to the hostan expression lowering amount of a ribozyme which cleaves RNA associatedwith expression of the gene. In another aspect, a method for treatingschizophrenia in a host is provided which includes lowering expressionof a gene selected from the group consisting of the gene encodingdecidual protein induced by progesterone (DEPP), the gene encodingadrenomedullin and the gene encoding cold shock domain protein A (csdA)by administering one or more nucleic acid molecules designed to promotetriple helix formation with said gene. In another aspect, a method fortreating schizophrenia is provided which includes reducing the amount ofavailable DEPP, adrenomedullin and/or csdA in a patient by administeringan effective amount of anti-DEPP, anti-adrenomedullin and/or anti-csdAantibody. In a preferred embodiment of the above aspects, the host is ahuman.

In another aspect, a method of screening for compounds which are usefulin the treatment of schizophrenia is provided which includes operativelylinking a reporter gene which expresses a detectable protein to aregulatory sequence for a gene selected from the group consisting ofDEPP, adrenomedullin and csdA to produce a reporter construct,transfecting a cell with the reporter construct, exposing thetransfected cell to a test compound, and comparing the level ofexpression of the reporter gene after exposure to the test compound tothe level of expression before exposure to the test compound, wherein alower level of expression after exposure is indicative of a compounduseful for the treatment of schizophrenia.

In another aspect, a transgenic nonhuman animal is provided which stablyincludes in its genome an increased copy number of a gene selected fromthe group consisting of the gene encoding decidual protein induced byprogesterone (DEPP), the gene encoding adrenomedullin and the geneencoding cold shock domain protein A (csdA) wherein the gene isexpressed at higher than baseline levels and the animal exhibitsabnormal behavior. In another aspect, a transgenic animal is providedwhich includes in its genome a gene selected from the group consistingof the gene encoding decidual protein induced by progesterone (DEPP),the gene encoding adrenomedullin and the gene encoding cold shock domainprotein A (csdA) wherein expression of the gene is enhanced by at leastone alteration in regulatory sequences of the gene such that the gene isexpressed at higher than baseline levels and the animal exhibitsabnormal behavior. In a preferred embodiment, the one or morealterations comprises substitution of a promoter having a higher rate ofexpression than the native promoter of the gene, in a more preferredembodiment the promoter is an inducible promoter. In another aspect, atransgenic nonhuman knockout animal is provided whose genome includes ahomozygous disruption in one or more genes selected from the groupconsisting of the gene encoding decidual protein induced by progesterone(DEPP), the gene encoding adrenomedullin and the gene encoding coldshock domain protein A (csdA), wherein said homozygous disruptionprevents the expression of the gene, and wherein said homozygousdisruption results in the transgenic knockout animal exhibitingdecreased expression levels of the one or more genes as compared to awild-type animal. In another aspect, the above transgenic nonhumananimals are utilized to screen for therapeutic agents that modulatesymptoms of schizophrenia by administering a candidate compound to thetransgenic nonhuman animals and determining the effect of the compoundon symptoms associated with schizophrenia. In a preferred embodiment ofthe above aspects, the transgenic nonhuman animal is a mammal.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a nucleic acid sequence encoding preproadrenomedullin.

FIG. 2 depicts a nucleic acid sequence encoding DEPP.

FIG. 3 depicts a nucleic acid sequence encoding cold shock domainprotein A (csdA).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present disclosure is based on the surprising discovery that threegenes, namely, the gene encoding decidual protein induced byprogesterone (DEPP), the gene encoding adrenomedullin and the geneencoding cold shock domain protein A (csdA) are associated withschizophrenia in affected individuals. More particularly, these threegenes are upregulated in the anterior cingulate of schizophrenicpatients as compared to normal patients. Accordingly, methods for thediagnosis, screening and evaluation of schizophrenia are provided inaccordance with the present invention. For example, assays fordetermination of increased levels of expression of the DEPP, csdA andadrenomedullin genes are provided. Moreover, nucleic acid moleculesencoding DEPP, csdA and adrenomedullin can be used as diagnostichybridization probes or used to design primers for diagnostic PCRanalysis for the identification of DEPP, csdA or adrenomedullin genemutations, allelic variations and regulatory defects in the DEPP, csdAor adrenomedullin genes. As used herein, “diagnosis” is intended togenerally apply to individuals while “screening” is generally applicableto populations or individuals. Both terms also encompass in vitromethods using for instance isolated tissue or isolated cells or cellsgrown in culture. The invention also encompasses antibodies to the DEPP,csdA and adrenomedullin gene products that can be used to decreaseavailable plasma levels of these proteins, as well as nucleotidesequences that can be used to inhibit gene expression (e.g., antisense,siRNA and ribozyme molecules), and gene or regulatory sequence bindingor replacement constructs designed to reduce or enhance gene expression(e.g., triple helix forming moieties or expression constructs that placethe genes under the control of a strong promoter system).

Adrenomedullin is a potent vasodilator peptide consisting of 52 aminoacids with the following sequence: YRQSMNNFQG LRSFGCRFGT CTVQKLAHQIYQFTDKDKDN VAPRSKISPQ GY (Seq. Id. No.1). The precursor, calledpreproadrenomedullin, is 185 amino acids long. It was originallydiscovered from a human pheochromocytoma, and belongs to the calcitoningene-related peptide (CGRP) family. cDNA encoding thepreproadrenomedullin protein is depicted in FIG. 1 (Seq. Id. No. 2).Adrenomedullin is produced and secreted by various types of cells, forexample, vascular endothelial and smooth muscle cells, cardiomyocytes,fibroblasts, macrophages, neurons, glial cells, and retinal pigmentepithelial cells. Expression of adrenomedullin is induced by hypoxia andproinflammatory cytokines. In addition to vasodilator actions, thispeptide has central inhibitory actions on water drinking and saltappetite, effects on the secretion of some hormones and cytokines,inotropic actions and effects on cell growth and apoptosis.Adrenomedullin is also produced by various non-endocrine tumors, as wellas endocrine tumors, and acts as a growth stimulatory factor for thetumor cells. Adrenomedullin seems to be involved in the pathophysiologyof many diseases, including ischemic heart diseases, inflammatorydiseases, tumors, and even eye diseases. Takahashi, Tohoku J. Exp. Med.,193 (2) 79-114 (2001). As used herein, the “gene encoding adrenomedulin”or “adrenomedulin gene” is meant to encompass preproadrenomedullin andadrenomedullin.

Decidual protein induced by progesterone (DEPP) consists of 212 aminoacids and has the following sequence: (Seq. Id. No. 3) 1 MRSRLLLSVAHLPTIRETTE EMLLGGPGQE PPPSPSLDDY VRSISRLAQP TSVLDKATAQ 61 GQPRPPHRPAQACRKGRPAV SLRDITARFS GQQPTLPMAD TVDPLDWLFG ESQEKQPSQR 121 DLPRRTGPSAGLWGPHRQMD SSKPMGAPRG RLCEARMPGH SLARPPQDGQ QSSDLRSWTF 181 GQSAQAMASRHRPRPSSVLR TLYSHLPVIH EL.A cDNA sequence encoding DEPP is shown in FIG. 2 (Seq. Id. No. 4). Thefunction of DEPP is currently unknown.

Cold shock domain protein A (csdA) consists of 372 amino acids and hasthe following sequence: (Seq. Id No. 5) 1 MSEAGEATTT TTTTLPQAPTEAAAAAPQDP APKSPVGSGA PQAAAPAPAA HVAGNPGGDA 61 APAATGTAAA ASLATAAGSEDAEKKVLATK VLGTVKWFNV RNGYGFINRN DTKEDVFVHQ 121 TAIKKNNPRK YLRSVGDGETVEFDVVEGEK GAEAANVTGP DGVPVEGSRY AADRRRYRRG 181 YYGRRRGPPR NYAGEEEEEGSGSSEGFDPP ATDRQFSGAR NQLRRPQYRP QYRQRRFPPY 241 HVGQTFDRRS RVLPHPNRIQAGEIGEMKDG VPEGAQLQGP VHRNPTYRPR YRSRGPPRPR 301 PAPAVGEAED KENQQATSGPNQPSVRRGYR RPYNYRRRPR PPNAPSQDGK EAKAGEAPTE 361 NPAPPTQQSS AE.csdA is a member of a family of transcriptional regulators. It isreported to bind and repress the promoter of GM-CSF. A cDNA sequenceencoding csdA (Seq. Id. No 6) is shown in FIG. 3.

The surprising expression characteristics of the DEPP, csdA andadrenomedullin genes were uncovered by examination of post mortemanterior cingulate samples from schizophrenic and normal subjects.Samples possessing high quality RNA were utilized for further study.Those skilled in the art are familiar with techniques which may beutilized to determine expression levels. For example, reversetranscriptase assays or DNA microarray analysis can be performedutilizing gene chip technology. Differentially expressed genes can beidentified using a number of methods developed in accordance withestablished principles. Statistical significance of the expressiondifferences between groups of samples may be determined utilizing thet-test, ANOVA or non-parametric tests. In accordance with the presentinvention, some genes were found to be upregulated in schizophrenicpatients while others were found to be downregulated compared tobaseline or normal levels. The terms “normal” and “baseline” are usedinterchangeably herein. Baseline levels are defined using conventionalstatistical techniques in connection with an analysis of a generalpopulation of non-schizophrenics. See, e.g., Example 1 herein. It shouldbe understood, in general, that methods not otherwise specified hereinare conducted in accordance with generally accepted principles known tothose skilled in the art.

Quantitative rtPCR (Q-PCR) may be conducted on the same samples used forthe expression level analysis described above. After conversion of RNAto cDNA using reverse transcriptase, although any conventional PCRtechnique can be utilized, a preferred technique may be based on theTaqMan® technique (Perkin Elmer Corp., Foster City, Calif.). Inconventional PCR assays, oligonucleotide primers are designedcomplementary to the 5′ and 3′ends of a DNA sequence of interest. Duringthermal cycling, DNA is heat denatured. The sample is then brought toannealing and extension temperatures in which the primers bind theirspecific complements and are extended by the addition of nucleotidetriphosphates by Taq polymerase. With repeated thermal cycling, theamount of template DNA is amplified. The presence of a dye, such asSybrGreen™, that fluoresces strongly when bound to DNA, allows the realtime monitoring of total amount of DNA product in the tube. By measuringthis signal, the amplified product can be quantified. The thresholdcycle (C_(T)) at which the fluorescent signal is measurably differentfrom the background noise is an accurate measure of the starting amountof cDNA in the tube and hence RNA in the sample. This method allows thequantitation of genes in a complex RNA by targeting specific DNAs. Ofthe genes initially identified by microarray analysis to bedifferentially expressed in schizophrenic patients, three, decidualprotein induced by progesterone (DEPP), csdA and adrenomedullin, wereshown to be differentially regulated In the original and a furtherindependent set of RNA samples.

In one aspect, a method of screening for schizophrenia in a populationis provided which includes determining, in members of the population,the magnitude of expression of a gene selected from the group consistingof the gene encoding decidual protein induced by progesterone (DEPP),the gene encoding adrenomedullin and the gene encoding cold shock domainprotein A (csdA) in a sample and comparing the magnitude of expressionto a baseline magnitude of expression of the gene, wherein increasedgene expression indicates the presence of schizophrenia. In anotheraspect, a method for diagnosing schizophrenia in a host is providedwhich includes determining the magnitude of expression of a geneselected from the group consisting of the gene encoding decidual proteininduced by progesterone (DEPP), the gene encoding adrenomedullin and thegene encoding cold shock domain protein A (csdA) in a sample andcomparing the magnitude of expression to a baseline magnitude ofexpression of the gene, wherein increased gene expression indicates thepresence of schizophrenia. In either of the the above screening ordiagnosing aspects, the sample may be taken, for example, from thebrain, spinal cord, lymphatic fluid, blood, urine or feces. In apreferred embodiment the sample is taken from the anterior cingulated.In another preferred embodiment, the population is human. There arenumerous techniques known to those with skill in the art to measure geneexpression in a sample. For example, RNA from a cell type or tissueknown, or suspected, to express the DEPP, csdA and/or adrenomedullingene, such as brain, may be isolated and tested utilizing hybridizationor PCR techniques such as are described above. The isolated RNA can bederived from primary cell culture from a patient or directly from abiological sample from a patient.

In another aspect, the present invention provides the use of a geneselected from the group consisting of the gene encoding decidual proteininduced by progesterone (DEPP), the gene encoding adrenomedullin and thegene encoding cold shock domain protein A (csdA) for the screening ofcompounds which are useful in the treatment of schizophrenia. Themethods entail identifying candidate or test compounds which binds DEPPor adrenomedullin or csdA and/or have a stimulatory or inhibitory effecton the activity or the expression of of DEPP or adrenomedullin or csdA.Preferably, the identification of candidate or test compounds isfollowed by further determining, which of the compounds that bind DEPPor adrenomedullin or csdA or have a stimulatory or inhibitory effect onthe activity or the expression of DEPP or adrenomedullin or csdA, havean effect on schizophrenia in an in vivo assay, such as for instance atransgenic animal as described by the present invention.

In one embodiment of such a detection scheme, a cDNA molecule issynthesized from an RNA molecule of interest (e.g., by reversetranscription of the RNA molecule into cDNA). A sequence within the cDNAis then used as the template for a nucleic acid amplification reaction,such as a PCR amplification reaction, or the like. The nucleic acidreagents used as synthesis initiation reagents (e.g., primers) in thereverse transcription and nucleic acid amplification steps of thismethod are chosen from among the DEPP, csdA or adrenomedullin genenucleic acid reagents. Those skilled in the art are familiar withtechniques for designing and obtaining suitable primers. See, e.g.,Table 1 in Example 2 below. The preferred lengths of such nucleic acidreagents are at least 9-30 nucleotides. For detection of the amplifiedproduct, the nucleic acid amplification may be performed usingradioactively or non-radioactively labeled nucleotides. Alternatively,enough amplified product may be made such that the product may bevisualized by standard ethidium bromide staining or by utilizing anyother suitable nucleic acid staining method.

Additionally, it is possible to perform such DEPP, csdA oradrenomedullin gene expression assays “in situ”, i.e., directly upontissue sections (fixed and/or frozen) of patient tissue obtained frombiopsies or resections, such that no nucleic acid purification isnecessary. Nucleic acid reagents such as those described above may beused as probes and/or primers for such in situ procedures.Alternatively, if a sufficient quantity of the appropriate cells can beobtained, standard Northern analysis can be performed to determine thelevel of mRNA expression of the DEPP, csdA or adrenomedullin genes.

Regardless of the method used to quantify the expression of the DEPP,csdA and/or adrenomedullin genes, the level of expression in a subjectof undefined etiology is compared to a known normal expression level. Ifthe expression level of one, or more than one, of these genes iselevated above the normal or baseline level by about 25%, a diagnosis ofschizophrenia may be made or confirmed. Determination of higher levelsmay be indicative of the severity of the disease.

As demonstrated by the Examples below, one technique for establishingbaseline levels may involve real time quantitative PCR. Those skilled inthe art are familiar with numerous techniques which may be utilized totest sample populations to obtain statistically sound results. Forexample, in carrying out this technique, a sample from a population ofnormal individuals is selected. The sample should be sufficientlydiverse in terms of age, sex, social status, geographical distribution,previous drug and medical histories, etc. and of sufficient size toprovide a meaningful statistical value. Thus, expression of the DEPP,csdA or adrenomedullin genes is measured in the sample of interest whichdefines distribution in the normal population. Baseline levels are thenassigned. A set of diseased subjects is also assayed to determinevalidity of the test by comparing results of the diseased sample tothose of the normal sample.

In accordance with the present invention, symptoms of DEPP, csdA and/oradrenomedullin gene mediated schizophrenia may be ameliorated bydecreasing the level of DEPP, csdA and/or adrenomedullin gene expressionand/or DEPP, csdA and adrenomedullin gene product activity by usingrespective DEPP, csdA and adrenomedullin gene sequences in conjunctionwith well-known antisense, siRNA, gene “knock-out,” ribozyme and/ortriple helix methods to decrease the level of DEPP, csdA and/oradrenomedullin gene expression. Among the compounds that may exhibit theability to modulate the activity, expression or synthesis of the DEPP,csdA and/or adrenomedullin gene, including the ability to ameliorate thesymptoms of a DEPP, csdA and/or adrenomedullin mediated schizophrenia,are antisense, ribozyme, and triple helix molecules. Such molecules maybe designed to reduce or inhibit either unimpaired, or d appropriate,mutant target gene activity. Techniques for the production and use ofsuch molecules are well known to those skilled in the art.

Antisense RNA and DNA molecules act to block the translation of mRNA byhybridizing to target mRNA and preventing protein translation. Antisenseapproaches involve the design of oligonucleotides that are complementaryto a target gene mRNA. The antisense oligonucleotides will bind to thecomplementary target gene mRNA transcripts and prevent translation.Absolute complementarity, although preferred, is not required.

Double-stranded RNA (dsRNA) can also be used to inhibit gene expressionby a mechanism generally known in the art as RNA interference (RNAi).RNAi is described for instance in U.S. Pat. No. 6,506,559 or in PatentApplications WO0244321 or WO0175164, the contents of which are herewithincorporated by reference. The length of the dsRNA is not crucial forRNAi according to the present invention, however, preferred dsRNAs aresuch which are generally known in the art as small-inhibitory RNAs(siRNAs). In a preferred embodiment, the siRNAs are short dsRNAs havinga length of 19 to 25 nucleotides. Most preferred are dsRNAs having alength of 21 to 23 nucleotides. The dsRNAs may be blunt ended or ligatedat or on at least one end with either loops composed of ribonucleotidesor deoxyribonucleotides or a chemical synthetic linker (WO00/44895). Ina preferred embodiment, the ribonucleic acid contains 3′-end nucleotideoverhangs on the antisense strand and/or the sense strands of the dsRNAof at least one ribonucleotide or deoxyribonucleotide, or modifiednucleotide. Preferred are overhangs with 1, 2, 3 or 4 nucleotides. Theoverhangs may contain both ribonucleotide(s) and deoxyribonucleotide(s)which in addition may contain modified sugar moietes. The overhang maybe of any sequence, but in a preferred embodiment, the overhang iscomplementary to the target mRNA strand. In another preferred embodimentthe overhang contains at least one UU group or dTdT group. In anotherpreferred embodiment, the overhang on the antisense strand has thepenultimate overhanging nucleotide complementary to the mRNA targetstrand. Preferably, such an overhang is a 2-nucleotides overhang. In afurther preferred embodiment, the overhang is composed of 4 Us. Inanother preferred embodiment, the extreme 3′-position of the siRNA is ahydroxyl group. Additionally, the 5′-end may be a hydroxyl or phosphategroup.

A sequence complementary” to a portion of an RNA, as referred to herein,means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex; in the case ofdouble-stranded antisense nucleic acids, a single strand of the duplexDNA may thus be tested, or triplex formation may be assayed. The abilityto hybridize will depend on both the degree of complementarity and thelength of the antisense nucleic acid. Generally, the longer thehybridizing nucleic acid, the more base mismatches with an RNA It maycontain and still form a stable duplex (or triplex, as the case may be).One skilled in the art can ascertain a tolerable degree of mismatch byuse of standard procedures to determine the melting point of thehybridized complex.

In one embodiment, oligonucleotides complementary to coding ornon-coding regions of the DEPP, csdA and/or adrenomedullin genes couldbe used in an antisense or RNAi approach to inhibit translation ofendogenous DEPP, csdA and/or adrenomedullin mRNA. Based upon thesequences presented in FIGS. 1-3 or upon allelic or homologous genomicand/or DNA sequences, one of skill in the art can easily choose andsynthesize any of a number of appropriate antisense or siRNA moleculesfor use in accordance with the present invention. Antisense nucleicacids should be at least six nucleotides in length, and are preferablyoligonucleotides ranging from 6 to about 50 nucleotides in length. Incertain preferred aspects the oligonucleotide length is from about 8 toabout 30 nucleotides.

Suitable antisense oligonucleotides herein encompass modifiedoligonucleotides which may exhibit enhanced stability, targeting orwhich otherwise exhibit enhanced therapeutic effectiveness. Examples ofmodified oligonucleotides include those where (1) at least twonucleotides are covalently linked via a synthetic internucleosidelinkage (i.e., a linkage other than a phosphodiester linkage between the5′ end of one nucleotide and the 3′ end of another nucleotide) and/or(2) a chemical group not normally associated with nucleic acids has beencovalently attached to the oligonucleotide. Examples of syntheticinternucleoside linkages are phosphorothioates, alkylphosphonates,phosphorodithioates, phosphate esters, alkylphosphonothioates,phosphoramidates, carbamates, phosphate triesters, acetamidates,peptides, and carboxymethyl esters. Modified oligonucleotides may alsohave covalently modified bases and/or sugars. For example,oligonucleotides having backbone sugars which are covalently attached tolow molecular weight organic groups other than a hydroxyl group at the3′ position and other than a phosphate group at the 5′ position. Thusmodified oligonucleotides may include a 2′-0-alkylated ribose group. Inaddition, modified oligonucleotides may include sugars such as arabinoseinstead of ribose. Modified oligonucleotides also can include baseanalogs such as C-5 propyne modified bases.

Antisense oligonucleotides or siRNA molecules may be synthesized bystandard techniques known in the art, e.g., by use of an automated DNAsynthesizer (such as are commercially available from Biosearch, AppliedBiosystems, etc.). As examples, phosphorothioate oligonucleotides may besynthesized by the method of Stein, et al. (1988, Nucl. Acids Res. 16,3209), methylphosphonate oligonucleotides can be prepared by use ofcontrolled pore glass polymer supports (Sarin, et al., 1988, Proc. Natl.Acad. Sci. U.S.A. 85, 7448-7451), etc.

While antisense nucleotides complementary to the target gene codingregion sequence could be used, those complementary to the transcribed,untranslated region are most preferred. A preferred site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Those with skill in the art are wellaware of various suitable initiation or termination codons in botheukaryotes and prokaryotes.

Antisense or siRNA molecules may be delivered to cells that express thetarget gene in vivo or in vitro. A number of methods have been developedfor delivering antisense DNA or RNA or siRNAi to cells; e.g., antisensemolecules can be injected directly into the tissue site, or modifiedantisense molecules, designed to target the desired cells (e.g.,antisense linked to peptides or antibodies that specifically bindreceptors or antigens expressed on the target cell surface) can beadministered systemically. A preferred technique involves constructing avector which incorporates a strong promoter to provide high expressionand good yield of antisense or siRNA oligonucleotides at the targetsite. The use of such a construct to transfect target cells in thepatient results in the transcription of sufficient amounts of singlestranded RNAs that will form complementary base pairs with theendogenous target gene transcripts and thereby prevent translation ofthe target gene mRNA or results in the transcription of sufficientamount of single-stranded RNAs complementary to each other that form adsRNA capable of inhibiting gene expression by RNAi. For example, avector can be introduced such that it is taken up by a cell and directsthe transcription of an antisense RNA. Such a vector can remain episomalor become chromosomally integrated, as long as it can be transcribed toproduce the desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods known to those in the art. Vectorscan be, e.g., plasmid, viral, or others typically used for replicationand expression in mammalian cells. It should be understood thatexpression of the sequence encoding the antisense RNA can be by anypromoter known in the art to act in mammalian, preferably human cells.Such promoters can be inducible or constitutive. Any type of plasmid,cosmid, YAC, BAC or viral vector can be used to prepare the recombinantDNA construct which can be introduced directly into the tissue site.Alternatively, viral vectors can be used that selectively infect thedesired tissue, in which case administration may be accomplished byanother route (e.g., systemically).

In related aspect, the present invention provides the use of on or moreantisense or siRNA molecules that specifically inhibit the expression ofDEPP, csdA and/or adrenomedullin genes for the manufacture of amedicament useful in the treatment of schizophrenia.

Ribozyme molecules designed to catalytically cleave target gene mRNAtranscripts can also be used to prevent or reduce translation of DEPP,csdA or adrenomedullin target gene mRNA and, therefore, expression oftarget gene product. (See, e.g., PCT International PublicationWO90/11364, published Oct. 4, 1990; Sarver, et: al., 1990, Science 247,1222-1225). Ribozymes are enzymatic RNA molecules capable of catalyzingthe specific cleavage of RNA: The mechanism of ribozyme action Involvessequence specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by an endonucleolytic cleavage event.The composition of ribozyme molecules must include one or more sequencescomplementary to the target gene mRNA, and must include the well knowncatalytic sequence responsible for mRNA cleavage. For this sequence,see, e.g., U.S. Pat. No. 5,093,246, incorporated herein by reference.

Ribozymes that cleave mRNA at site specific recognition sequences can beused to destroy target gene mRNAs. For example, hammerhead ribozymes maybe utilized to cleave mRNAs at locations dictated by flanking regionsthat form complementary base pairs with the target mRNA. The solerequirement is that the target mRNA have the following sequence of twobases: 5′-UG-3′. The construction and production of hammerhead ribozymesis well known in the art. Preferably, the ribozyme is engineered so thatthe cleavage recognition site is located near the 5′ end of the targetgene mRNA, i.e., to increase efficiency and minimize the intracellularaccumulation of non-functional protein fragments. Suitable ribozymesalso include RNA endoribonucleases such as the one that occurs naturallyin Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA). Thistype of ribozymes have an eight base pair active site which hybridizesto a target RNA sequence to effect cleavage of the target RNA.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g., for improved stability, targeting, etc.) andshould be delivered to cells that express the target gene in vivo. Apreferred method of delivery involves using a DNA construct “encoding”the ribozyme under the control of a strong constitutive promoter, sothat transfected cells will produce sufficient quantities of theribozyme to destroy endogenous DEPP csdA and/or adrenomedullin genemessages and inhibit translation. Because ribozymes, unlike antisensemolecules, are catalytic, a lower intracellular concentration isrequired for efficiency.

Alternatively, endogenous DEPP, csdA and/or adrenomedullin geneexpression can be reduced by targeting deoxyribonucleotide sequencescomplementary to the regulatory region of the target DEPP, csdA and/oradrenomedullin genes (i.e., the target gene promoter and/or enhancers)to form triple helical structures that prevent transcription of thetarget gene in target cells in the body. Nucleic acid molecules to beused in triplex helix formation for the inhibition of transcriptionshould be single stranded and composed of deoxynucleotides. The basecomposition of these oligonucleotides must be designed to promote triplehelix formation via Hoogsteen base pairing rules, which generallyrequire sizeable stretches of either purines or pyrimidines to bepresent on one strand of a duplex. Nucleotide sequences may bepyrimidine-based, which will result in TAT and CGC⁺ triplets across thethree associated strands of the resulting triple helix. Thepyrimidine-rich molecules provide base complementarity to a purine-richregion of a single strand of the duplex in a parallel orientation tothat strand. In addition, nucleic acid molecules may be chosen that arepurine-rich, for example, contain a stretch of G residues. Thesemolecules will form a triple helix with a DNA duplex that is rich in GCpairs, in which the majority of the purine residues are located on asingle strand of the targeted duplex, resulting in GGC triplets acrossthe three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so-called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′, 3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizeable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

In related aspect, the present invention provides the use of on or moreribozyme or nucleic acid molecule promoting triple helix formation thatspecifically inhibit the expression of DEPP, csdA and/or adrenomedullingenes for the manufacture of a medicament useful in the treatment ofschizophrenia.

Anti-sense RNA and DNA, siRNA, ribozyme, and triple helix moleculesdescribed herein may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules, as discussed above. These includetechniques for chemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors that incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

In another aspect, the present Invention provides the use of a geneselected from the group consisting of the gene encoding decidual proteininduced by progesterone (DEPP), the gene encoding adrenomedullin and thegene encoding cold shock domain protein A (csdA) as a biomarker forschizophrenia. In addition, this invention provides methods forscreening a subject to determine if biomarkers for schizophrenia arepresent in order to confirm a diagnosis made on purely clinical grounds.This invention also provides methods for monitoring the severity orprogression of the schizophrenia in an individual.

A method of modulating DEPP, csdA and/or adrenomedullin to treatschizophrenia is provided by exposing neutralizing antibodies to DEPP,csdA and/or adrenomedullin proteins. By providing for controlledexposure to such antibodies, protein abundances/activities can becontrollably modified. For example, antibodies to suitable epitopes onprotein surfaces may decrease the abundance, and thereby indirectlydecrease the activity, of the wild-type active form of DEPP, csdA and/oradrenomedullin proteins by aggregating active forms into complexes withless or minimal activity as compared to the wild-type unaggregatedwild-type form. Alternately, antibodies may directly decrease proteinactivity by, e.g., interacting directly with active sites or by blockingaccess of substrates to active site. In either case, antibodies can beraised against specific protein species and their effects screened. Theeffects of the antibodies can be assayed and suitable antibodiesselected that lower the target protein species concentration and/oractivity. Such assays involve introducing antibodies into a cell orsurrounding media, and assaying the concentration of the wild-typeamount or activities of the target protein by standard means (such asimmunoassays) known in the art. The net activity of the wild-type formcan be assayed by assay means appropriate to the known activity of thetarget protein.

Thus, in another aspect, the present invention provides the use of anantibody or several antibodies that specifically bind an epitope ofDEPP, csdA and/or adrenomedullin proteins for the manufacture of amedicament useful in the treatment of schizophrenia.

Antibodies can be introduced into cells in numerous ways, including, forexample, microinjection of antibodies into a cell (Morgan et al., 1988,Immunology Today 9:84-86) or transforming hybridoma mRNA encoding adesired antibody into a cell (Burke et al., 1984, Cell 36:847-858). In afurther technique, recombinant antibodies can be engineered andectopically expressed in a wide variety of non-lymphoid cell types tobind to target proteins as well as to block target protein activities.Preferably, expression of the antibody is under control of acontrollable promoter, such as the Tet promoter. A first step is theselection of a particular monoclonal antibody with appropriatespecificity to the target protein. Then sequences encoding the variableregions of the selected antibody can be cloned into various engineeredantibody formats, including, for example, whole antibody, Fab fragments,Fv fragments, single chain Fv fragments (VH and VL regions united by apeptide linker) (“ScFv” fragments), diabodies (two associated ScFvfragments with different specificities), and so forth. Intracellularlyexpressed antibodies of the various formats can be targeted intocellular compartments by expressing them as fusions with the variousknown intracellular leader sequences.

Methods for the production of antibodies capable of specificallyrecognizing one or more DEPP, csdA and or adrenomedullin gene productepitopes or epitopes of conserved variants or peptide fragments of theDEPP, csdA and/or adrenomedullin gene products are well known in theart. Such antibodies may include, but are not limited to, polyclonalantibodies, monoclonal antibodies (mAbs), humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab′)₂ fragments,fragments produced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above.

Such antibodies may also be used, for example, in the detection of aDEPP, csdA and/or adrenomedullin gene product in an biological sampleand may, therefore, be utilized as part of a diagnostic or prognostictechnique whereby patients may be tested for abnormal levels of DEPP,csdA and/or adrenomedullin gene products, and/or for the presence ofabnormal forms of such gene products. Such antibodies may also beutilized in conjunction with, for example, compound screening schemes,for the evaluation of the effect of test compounds on DEPP, csdA and/oradrenomedullin gene product levels and/or activity.

For the production of antibodies against a DEPP, csdA and/oradrenomedullin gene product, various host animals may be immunized byinjection with a DEPP, csdA and/or adrenomedullin gene product, or aportion thereof. Such host animals may include, but are not limited torabbits, mice, and rats, to name but a few. Various adjuvants may beused to increase the immunological response, depending on the hostspecies, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as a DEPP, csdA and/or adrenomedullin gene product, or an antigenicfunctional derivative thereof. For the production of polyclonalantibodies, host animals such as these described above, may be immunizedby injection with DEPP, csdA and/or adrenomedullin supplemented withadjuvants as also described above.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique that providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256, 495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4, 72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80, 2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb may be cultivated in vitro or in vivo.

In addition, techniques developed for the production of “chimericantibodies” (Morrison, et al., 1984, Proc. Natl. Acad. Sci., 81,6851-6855; Neuberger, et al., 1984, Nature 312, 604-608; Takeda, et al.,1985, Nature, 314, 452-454) by splicing the genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Techniques have also been developed for the production ofhumanized antibodies. (See, e.g., Queen, U.S. Pat. No. 5,585,089,). Animmunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions, referredto as complementarity determining regions (CDRs). The extent of theframework region and CDRs have been precisely defined (see, e.g.,“Sequences of Proteins of Immunological Interest”, Kabat, E. et al.,U.S. Department of Health and Human Services (1983)). Briefly, humanizedantibodies are antibody molecules from non-human species having one ormore CDRs from the non-human species and a framework region from a humanimmunoglobulin molecule. Alternatively, techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778; Bird,1988, Science 242, 423-426; Huston, et al., 1988, Proc. Natl. Acad. Sci.USA 85,5879-5883; and Ward, et al., 1989, Nature 334, 544-546) can beadapted to produce single chain antibodies against DEPP, csdA and/oradrenomedullin gene products. Single chain antibodies are formed bylinking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab′)₂ fragments, which can be produced by pepsindigestion of the antibody molecule and the Fab fragments, which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse, etal., 1989, Science, 246, 1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.

Antibodies, or fragments of antibodies, such as those described, above,may be used to quantitatively or qualitatively detect the presence ofDEPP, csdA and/or adrenomedullin gene products or conserved variants orpeptide-fragments thereof. This can be accomplished, for example, byimmunofluorescence techniques employing a fluorescently labeled antibodycoupled with light microscopic, flow cytometric, or fluorometricdetection.

The antibodies (or fragments thereof) useful in the present inventionmay be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of DEPP, csdA and/oradrenomedullin gene products, conserved variants or peptide fragmentsthereof. In situ detection may be accomplished by removing ahistological specimen from a patient, and applying thereto a labeledantibody that binds to a DEPP, csdA and/or adrenomedullin polypeptide.The antibody (or fragment) is preferably applied by overlaying thelabeled antibody (or fragment) onto a biological sample. Through the useof such a procedure, it is possible to determine not only the presenceof DEPP, csdA and/or adrenomedullin, conserved variants or peptidefragments, but also its distribution in the examined tissue. Using thepresent invention, those of ordinary skill will readily recognize thatany of a wide variety of histological methods (such as stainingprocedures) can be modified in order to achieve in situ detection ofDEPP, csdA and/or adrenomedullin.

Immunoassays for DEPP, csdA and/or adrenomedullin, conserved variants,or peptide fragments thereof will typically comprise incubating asample, such as a biological fluid, a tissue extract, freshly harvestedcells, or lysates of cells in the presence of a detectably labeledantibody capable of identifying DEPP, csdA and/or adrenomedullin,conserved variants or peptide fragments thereof, and detecting the boundantibody by any of a number of techniques well-known in the art. Thebiological sample may be brought in contact with and immobilized onto asolid phase support or carrier, such as nitrocellulose, that is capableof immobilizing cells, cell particles or soluble proteins. The supportmay then be washed with suitable buffers followed by treatment with thedetectably labeled DEPP, csdA and/or adrenomedullin specific antibodies.The solid phase support may then be washed with the buffer a second timeto remove unbound antibody. The amount of bound label on the solidsupport may then be detected by conventional means.

One of the ways in which the DEPP, csdA and/or adrenomedullin-specificantibodies can be detectably labeled is by linking the same to anenzyme, such as for use in an enzyme immunoassay (EIA). The enzyme,which is bound to the antibody, will react with an appropriatesubstrate, preferably a chromogenic substrate, in such a manner as toproduce a chemical moiety that can be detected, for example, byspectrophotometric, fluorimetric or by visual means. Enzymes that can beused to detectably label the antibody are well known. The detection canbe accomplished by colorimetric methods that employ a chromogenicsubstrate for the enzyme. Detection may also be accomplished by visualcomparison of the extent of enzymatic reaction of a substrate incomparison with similarly prepared standards. Detection may also beaccomplished using any of a variety of other immunoassays. For example,by radioactively labeling the antibodies or antibody fragments, it ispossible to detect DEPP, csdA and/or adrenomedullin through the use of aradioimmunoassay (RIA). The radioactive isotope can be detected by suchmeans as the use of a gamma counter or a scintillation counter or byautoradiography. It is also possible to label the antibody with afluorescent compound. When the fluorescently labeled antibody is exposedto light of the proper wavelength, its presence can then be detected dueto fluorescence. Among the most commonly used fluorescent labelingcompounds are green fluorescent protein, fluorescein isothiocyanate,rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine. The antibody can also be detectably labeled usingfluorescence emitting metals such as ¹⁵²Eu, or others of the lanthanideseries. These metals can be attached to the antibody using such metalchelating groups as diethylenetriaminepentacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA). The antibody also can bedetectably labeled by coupling it to a chemiluminescent compound. Thepresence of the chemiluminescent-tagged antibody is then determined bydetecting the presence of luminescence that arises during the course ofa chemical reaction. Examples of particularly useful chemiluminescentlabeling compounds are luminol, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester. Likewise, a bioluminescentcompound may be used to label the antibody of the present invention.Bioluminescence is a type of chemiluminescence found in biologicalsystems in which a catalytic protein increases the efficiency of thechemiluminescent reaction. The presence of a bioluminescent protein isdetermined by detecting the presence of luminescence. Importantbioluminescent compounds for purposes of labeling include luciferin,luciferase and aequorin.

The present invention contemplates production of animal models that haveabnormal expression levels of DEPP, csdA and/or adrenomedullin to studythe effects of increased or decreased levels of these proteins on suchanimals. Such animals provide test subjects for determining the effectsof therapeutic or potentially therapeutic compounds on schizophrenia.Accordingly, the DEPP, csdA and/or adrenomedullin gene products can beexpressed in transgenic animals. Animals of any species, including, butnot limited to, mice, rats, rabbits, guinea pigs, pigs, mini-pigs,goats, sheep, and non-human primates, e.g., baboons, monkeys, andchimpanzees may be used to generate DEPP, csdA and/or adrenomedullintransgenic animals. The term “transgenic,” as used herein, refers toanimals expressing DEPP, csdA and/or adrenomedullin gene sequences froma different species (e.g., mice expressing human DEPP, csdA and/oradrenomedullin gene sequences), as well as animals that have beengenetically engineered to overexpress endogenous (i.e., same species)DEPP, csdA and/or adrenomedullin sequences or animals that have beengenetically engineered to no longer express endogenous DEPP, csdA and/oradrenomedullin gene sequences (i.e., “knockout” animals), and theirprogeny.

Any technique known in the art may be used to introduce DEPP, csdAand/or adrenomedullin genes into animals to produce the founder lines oftransgenic animals. Such techniques include, but are not limited topronuclear microinjection (Hoppe and Wagner, 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten, et al., 1985, Proc. Natl. Acad. Sci., USA 82, 6148-6152); genetargeting in embryonic stem cells (Thompson, et al., 1989, Cell 56,313-321); electroporation of embryos (Lo, 1983, Mol. Cell. Biol. 3,1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57, 717-723) (For a review of such techniques, see Gordon, 1989,Transgenic Animals, Intl. Rev. Cytol. 115, 171-229). Any technique knownin the art may be used to produce transgenic animal clones containing aDEPP, csdA and/or adrenomedullin transgene, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal or adult cells induced to quiescence (Campbell, et al., 1996,Nature 380, 64-66; Wilmut, et al., 1997,Nature 385, 810-813).

The present invention provides for transgenic animals that carry anDEPP, csdA and/or adrenomedullin transgene in all their cells, as wellas animals that carry the transgene in some, but not all their cells,i.e., mosaic animals. The transgene may be integrated as a singletransgene or in concatamers, e.g., head-to-head tandems or head-to-tailtandems. The transgene may also be selectively introduced Into andactivated in a particular cell type by following, for example, theteaching of Lasko et al. (Lasko, et al., 1992, Proc. Natl. Acad. Sci.USA 89, 6232-6236). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the DEPP, csdA and/or adrenomedullin transgene beintegrated into the chromosomal site of the endogenous DEPP, csdA and/oradrenomedullin gene, gene targeting is preferred. Briefly, when such atechnique is to be utilized, vectors containing some nucleotidesequences homologous to the endogenous DEPP, csdA and/or adrenomedullingene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous DEPP, csdA and/oradrenomedullin gene. The transgene may also be selectively introducedinto a particular cell type, thus inactivating the endogenous DEPP, csdAand/or adrenomedullin gene in only that cell type, by following, forexample, the teaching of Gu, et al., 1994, Science 265,103-106. Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

As mentioned above, transgenic knockout animals are also providedherein. In such transgenic animals DEPP, csdA and/or adrenomedullin geneexpression is undetectable or insignificant. Any technique known in theart may be used to produce such transgenic knockout animals. This may beachieved by a variety of mechanisms, e.g., alteration of any or all ofthe DEPP, csdA and/or adrenomedullin genes by, e.g., introduction of adisruption of the coding sequence, e.g., insertion of one or more stopcodons, insertion of a DNA fragment, etc., deletion of regulatory orcoding sequence, substitution of stop codons for coding sequence, etc.The transgenic animals may be either homozygous or heterozygous for thealteration. A functional knock-out may also be achieved by theintroduction of an anti-sense construct that blocks expression of thenative genes. Knockouts also include conditional knockouts such as wherealteration of the target gene occurs upon exposure of the animal to asubstance that promotes target gene alteration, introduction of anenzyme that promotes recombination at the target gene site, or othermethod for directing the target gene alteration postnatally.

Once transgenic animals have been generated, the expression of therecombinant DEPP, csdA and/or adrenomedullin gene may be assayedutilizing standard techniques. Initial screening may be accomplished bySouthern blot analysis or PCR techniques to analyze animal tissues toassay whether integration of the transgene has taken place. The level ofmRNA expression of the transgene in the tissues of the transgenicanimals may also be assessed using techniques described above and thosethat include but are not limited to Northern blot analysis of tissuesamples obtained from the animal, in situ hybridization analysis, andRT-PCR (reverse transcriptase PCR). Samples of DEPP, csdA and/oradrenomedullin gene-expressing tissue, may also be evaluatedimmunocytochemically using antibodies specific for the DEPP, csdA and/oradrenomedullin transgene product.

Through use of the subject transgenic animals or cells derivedtherefrom, one can identify ligands or substrates that modulatephenomena associated with schizophrenia, e.g., behavioral phenomena. Awide variety of assays may be used for this purpose, includingbehavioral studies, determination of the localization of drugs afteradministration and the like. Depending on the particular assay, wholeanimals may be used, or cells derived therefrom. Cells may be freshlyisolated from an animal, or may be immortalized in culture. Cells ofparticular interest are derived from neural tissue.

The term “therapeutic agent” as used herein describes any molecule, e.g.protein, carbohydrate, metal or organic compound, with the capability ofaffecting the molecular and clinical phenomena associated withschizophrenia. Generally a plurality of assay mixtures may be run inparallel with different agent concentrations to obtain a differentialresponse to the various concentrations. Typically, one of theseconcentrations serves as a negative control, i.e. at zero concentrationor below the level of detection.

Candidate therapeutic agents encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 50 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate therapeutic agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Candidatetherapeutic agents are also found among biomolecules Including, but notlimited to: peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.

As mentioned above, antibodies specific for DEPP, csdA and/oradrenomedullin gene products may be used in screening immunoassays,particularly to detect the level of such products in a cell or sample.The number of cells in a sample will generally be at least about 10³,usually at least 10⁴ more usually at least about 10⁵. The cells may bedissociated, in the case of solid tissues, or tissue sections may beanalyzed. Alternatively a lysate of the cells may be prepared. Forexample, detection may utilize staining of cells or histologicalsections, performed in accordance with conventional methods. Theantibodies of interest are added to the cell sample, and incubated for aperiod of time sufficient to allow binding to the epitope, usually atleast about 10 minutes. The antibody may be labeled with radioisotopes,enzymes, fluorescers, chemiluminescers, or other labels for directdetection. Alternatively, a second stage antibody or reagent is used toamplify the signal. Such reagents are well known in the art For example,the primary antibody may be conjugated to biotin, with horseradishperoxidase-conjugated avidin added as a second stage reagent. Finaldetection uses a substrate that undergoes a color change in the presenceof the peroxidase. The absence or presence of antibody binding may bedetermined by various methods, including flow cytometry of dissociatedcells, microscopy, radiography, scintillation counting, etc.

A number of assays are known in the art for determining the effect of adrug on animal behavior and other phenomena associated withschizophrenia. Some examples are provided, although it will beunderstood by one of skill in the art that many other assays may also beused. The subject animals may be used by themselves, or in combinationwith control animals.

The screen using the transgenic animals of the invention can employ anyphenomena associated with schizophrenia that can be readily assessed inan animal model. The screening for schizophrenia can include assessmentof phenomena including, but not limited to: 1) analysis of molecularmarkers (e.g., levels of expression of DEPP, csdA and/or adrenomedullingene products in brain tissue; presence/absence in brain tissue ofvarious DEPP, csdA and/or adrenomedullin splice variants; 2) assessmentof behavioral symptoms associated with memory and learning; and 3)detection of neurodegeneration. Preferably, the screen will includecontrol values (e.g., the level of DEPP, csdA and/or adrenomedullinproduction in the test animal In the absence of test compound(s)). Testsubstances which are considered positive, I.e., likely to be beneficialin the treatment of schizophrenia, will be those which have asubstantial effect upon a schizophrenia associated phenomenon (e.g.,test agents that are able to normalize erratic or abnormal behavior orthat reduce the level of DEPP, csdA and/or adrenomedullin production towithin the normal range).

The present invention also encompasses the use of cell-based assays orcell-lysate assays (e.g., in vitro transcription or translation assays)to screen for compounds or compositions that modulate DEPP, csdA and/oradrenomedullin gene expression. To this end, constructs containing areporter sequence linked to a regulatory element of the any or all ofthe genes encoding DEPP, csdA and/or adrenomedullin can be used inengineered cells, or in cell lysate extracts, to screen for compoundsthat modulate the expression of the reporter gene product at the levelof transcription. For example, such assays could be used to identifycompounds that modulate the expression or activity of transcriptionfactors involved in expression of the genes encoding DEPP, csdA and/oradrenomedullin, or to test the activity of triple helix polynucleotides.Alternatively, engineered cells or translation extracts can be used toscreen for compounds (including antisense and ribozyme constructs) thatmodulate the translation of DEPP, csdA and/or adrenomedullin mRNAtranscripts, and therefore, affect expression of the DEPP, csdA and/oradrenomedullin gene products. Thus, regulatory regions such as apromoter are operatively linked to a gene encoding a reporter moleculesuch as green fluorescent protein (GFP), luciferase and the like, tocreate a reporter construct which is regulated by the DEPP, csdA and/oradrenomedullin regulatory sequences. The gene construct is thentransfected into a desired cell such as a neuronal cell. The baselineexpression levels of the reporter molecule are then calculated usingconventional methods. The cell is then exposed to a test compound andthe level of expression of the reporter molecule is determined andcompared to the baseline levels. A compound which reduces the amount ofreporter expression is a candidate for the treatment of schizophrenia. Asecond screening procedure may then be instituted to determine whetherthe compound affects the level of expression of the gene(s) encodingDEPP, csdA and/or adrenomedullin by measuring the amount of RNA orprotein from the native gene(s). Construction of neuronal cellsincorporating a reporter gene for determining the effect of compounds onexpression is known, e.g., see, Asselbergs et al., Nucleic Acids Res27:1826-33(1998), incorporated herein by reference.

Antisense compounds, ribozymes, antibodies and other DEPP, csdA and/oradrenomedullin knockout devices or modulators (collectively referred tofor convenience as the “modulators”) described herein may be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecular structures or mixtures of compounds, as for example,liposomes, receptor targeted molecules, oral, rectal, topical, or otherformulations, for assisting in uptake, distribution and/or absorption.Those skilled in the art are familiar with a myriad of techniques toproduce such devices.

It is contemplated that the modulators may encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents. The term“pharmaceutically acceptable salts” refers to physiologically andpharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undue toxicological effects thereto. Suchcompounds may be prepared according to conventional methods by one ofskill in the art. (Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The term “prodrug” indicates a therapeutic agentthat is prepared in an inactive form that is converted to an active form(i.e., drug) within the body or cells thereof by the action ofendogenous enzymes or other chemicals and/or conditions. In particular,prodrug versions of the oligonucleotides may be prepared as SATE[(S-acetyl-2-thioethyl)phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., or in WO 94/26764 to Imbachet al.

The modulators herein can be utilized for diagnostics, therapeutics,prophylaxis and as research reagents and kits. For therapeutics, ananimal, preferably a human, suspected of having a schizophrenic diseaseor disorder which can be treated by modulating the expression of one ormore DEPP, csdA and/or adrenomedullin regulated genes, is treated byadministering modulators in accordance with this invention. Themodulators can be utilized In pharmaceutical compositions by adding aneffective amount of one or more modulators to a suitablepharmaceutically acceptable diluent or carrier. Those skilled in the artare familiar with numerous techniques and formulations utilized tocompound pharmaceutical compositions. The pharmaceutical compositions ofthe present invention may be administered in a number of ways dependingupon whether local or systemic treatment is desired and upon the area tobe treated. Administration may be topical (including ophthalmic and tomucous membranes including vaginal and rectal delivery), pulmonary,e.g., by inhalation or insufflation of liquids, powders or aerosols,including by nebulizer; intratracheal, intranasal, enteral, epidermaland transdermal), oral, sublingual, buccal or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; intramedullaryor intracranial, e.g., intrathecal or intraventricular, administration.Oligonucleotides with at least one 2′-O-methoxyethyl modification may beuseful for oral administration.

Pharmaceutical compositions for topical administration may includetransdermal patches, ointments, lotions, creams, gels, drops,suppositories, sprays, liquids and powders. Conventional pharmaceuticalcarriers, aqueous, powder or oily bases, thickeners and the like may benecessary or desirable. Compositions and formulations for oraladministration include powders or granules, suspensions or solutions inwater or non-aqueous media, capsules, sachets, troches or tablets.Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids orbinders may be desirable. Compositions for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, suspensions, foams andliposome-containing formulations. These compositions may be generatedfrom a variety of components that include, but are not limited to,preformed liquids, self-emulsifying solids and self-emulsifyingsemisolids, according to conventional methods, by one of skill in theart.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product. Further details on techniques for formulation andadministration of numerous dosage forms may be found in the latestedition of Remington's Pharmaceutical Sciences (Maack Publishing Co.,Easton, Pa.). The compositions may be administered alone or incombination with at least one other agent, such as stabilizing compound,which may be administered In any sterile, biocompatible pharmaceuticalcarrier, including, but not limited to, saline, buffered saline,dextrose, and water. The compositions may be administered to a patientalone, or in combination with other agents, drugs or hormones.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances that increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers may also be used for delivery. Optionally, the suspensionmay also contain suitable stabilizers or agents that increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions. For topical or nasal administration, penetrantsappropriate to the particular barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the modulators are contained in an effective amountto achieve the intended purpose. The determination of an effective doseis well within the capability of those skilled in the art. For anycompound, the therapeutically effective dose can be estimated initiallyeither in cell culture assays, e.g., of neoplastic cells, or in animalmodels, usually mice, rabbits, dogs, or pigs. The animal model may alsobe used to determine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans. A therapeuticallyeffective dose refers to that amount of active Ingredient, whichameliorates, partially or completely, the symptoms or condition.Therapeutic efficacy and toxicity may be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., ED50 (the dose therapeutically effective in 50% of the population)and LD50 (the dose lethal to 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index, and itcan be expressed as the ratio, LD50/ED50. Pharmaceutical compositionsthat exhibit large therapeutic indices are preferred. The data obtainedfrom cell culture assays and animal studies is used in formulating arange of dosage for human use. The dosage contained in such compositionsis preferably within a range of circulating concentrations that includethe ED50 with little or no toxicity. The dosage varies within this rangedepending upon the dosage form employed, sensitivity of the patient, andthe route of administration.

The exact dosage will be determined by the practitioner in light offactors related to the subject that require treatment. Dosage andadministration are adjusted to provide sufficient levels of themodulators to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation. Normal dosage amounts may vary from 0.1to 100,000 micrograms, up to a total dose of about 1 g per kilogram,depending upon the route of administration. Guidance as to particulardosages and methods of delivery is provided in the literature andgenerally available to practitioners in the art. Those skilled in theart will employ different formulations for nucleotides than for proteinsor their inhibitors. Similarly, delivery of polynucleotides orpolypeptides will be specific to particular cells, conditions,locations, etc.

All references cited herein are incorporated by reference in theirentireties. The following examples are included for purposes ofillustration and should not be construed as limiting the presentinvention.

EXAMPLE 1 DNA Microarray Analysis

Human anterior cingulate samples are obtained from 10 normal and 10schizophrenic deceased subjects (Maryland Psychiatric Research Clinic,Baltimore, Md.). Good quality RNA is obtained from 9 normal (“N1”) and 7schizophrenic (“S1”) samples.

The microarray analysis is performed essentially as follows. Briefly, 5μg or less total RNA is used to synthesize cDNA which is then used as atemplate to generate biotinylated cRNA. 15 to 30 μg labeled RNA isobtained and hybridized to Affymetrix (Santa Clara, Calif.) the HumanGenome U95Av2 Array of the GeneChip® Human Genome U95 Set (HG-U95Av2contains ≈12,000 sequences of full length genes) in accordance with theprotocols found in the GeneChip® technical manual. Each sample isprofiled in duplicate. After sample hybridization, microarrays arewashed and scanned with a laser scanner.

The images obtained are used to generate absolute text files foranalysis using Affymetrix GeneChip® Gene Expression Analysis Algorithmsversion 4. Differentially expressed genes between the normal andschizophrenic derived samples are ranked using a pattern recognitionalgorithm developed in accordance with established principles whichgenerated a score for each gene being compared. The following threeconditions are required for a score (equal to the mean fold change) tobe generated: (1) t-test p-value<0.5%; (2) average fold-change>1.5; (3)maximum mean AvgDiff (expression levels on an Affymetrix chip)>200. Ifone or more of the above conditions is not met by a gene in comparison,the score assigned is zero. Several candidate genes are found to bedifferentially expressed in schizophrenic patients when compared tonormal.

EXAMPLE 2 Real Time Quantitative PCR Confirmation of DifferentiallyRegulated Genes

Probe pairs for real time quantitative PCR (Q-PCR) are designed for 38altered genes identified in Example 1. Affymetrix provides a file ofsequences from which the probes on the chip are derived. From this file,the sequences corresponding to these 38 altered genes are obtained, andthe probe pairs are prepared. Where a good pair of primers cannot beobtained from Affymetrix sequence, a longer sequence can be obtainedfrom Ref Seq. (See Pruitt K D, Maglott D R Nucleic Acids Res Jan. 1,2001;29(1):137-140;Introducing RefSeq and LocusLink: curated humangenome resources at the NCBl., Pruitt K D, Katz K S, Sicotte H, MaglottD R Trends Genet. January 2000;16(1):44-47) with a good BLAST scoreagainst the Affymetrix sequence and the primers are designed from thatsequence. The sequences of the probe pairs are presented in Table 1.TABLE 1 Affymetrix Primer Gene ID Primer name Sequence 5′-3′ nameSequence 5′-3′ GAPDH GAPDH5F CAGGGCTGCTTTTAACTCTGGTA GAPDH5RGGGTGGAATCATATTGGAACATGT (Seq. Id. 7) (Seq. Id. 8) 1622_at 1622p2FTTTTAATCTCTCGACTGAATGGACTTT 1622p2R CACAACTCATCCCCTCTTGTGTAC (Seq. Id.9) (Seq. Id. 10) 1731_at 1731for TGTGTTTTCAGCAAATTCCAGATT 1731revTGTCATCATAAAAATAGAAAGTAGG (Seq. Id. 11) AAAGATT (Seq. Id. 12) 32279_at32279for TTCTGTTCTCAAATGCTAAATGCAA 32279rev GGGAATGTTGATGTCATTCAGAAA(Seq. Id. 13) (Seq. Id. 14) 32757_at 32757for GCCTGTTGCAGAGTTTTTCTGTAA32757rev TTCCAATATTCTCTAACACGTACAC (Seq. Id. 15) (Seq. Id. 16) 32814_at32814for GGTAGAAGAAACAATGCAAGACATACAT 32814rev CCCTTGTTAATGATGCCTGTTCTAT(Seq. Id. 17) (Seq. Id. 18) 33372_at 33372forGGAAATGTACCTGAAAAGGATTTTAGA 33372rev ACTACATCCCCTCCATGTGCAT (Seq. Id.19) (Seq. Id. 20) 33890_at 33890for AGTGCAGATTTATACTCCTGACGTGT 33890revCAAGACTTATAATCATGAAATACAG (Seq. Id. 21) AATTAAMGTT (Seq. Id. 22)34250_at 34250for TGTCCGTCCCTGTTTTTGCT 34250rev CACCCTGCCTTTCCCTAGAGA(Seq. Id. 23) (Seq. Id. 24) 34375_AT 34375for GGAAGATCTCAGTGCAGAGGCT34375rev GATCTCCTTGGCCACAATGGT (Seq. Id. 25) (Seq. Id. 26) 34582_at34582for TGGATGGAGGACAGATTGTGACT 34582rev AATGAGGAGCATGGTGACCAG (Seq.Id. 27) (Seq. Id. 28) 34777_AT 34777for TCGCCCACAAACTGATTTCTC 34777revACGCATTGCACTTTTCCTCTTT (Seq. Id. 29) (Seq. Id. 30) 35109_at 35109forCCCTCCTGCTGTGCTCTTCT 35109rev GTGACAGACACACATCAGCCACTA (Seq. Id. 31)(Seq. Id. 32) 35680_r_at 35680_rfor GATAGAATTAACTCGTATTTTTCTATGG35680_rrev GGAGAAAGCCAAGGGAAACAA TTTTAA (Seq. Id. 33) (Seq. Id. 34)35857_at 35857for TCCTTACTTGTCATCAGAGACGAACT 35857revAAAAATCTGTAGGGAATGCATCCTT (Seq. Id. 35) (Seq. Id. 36) 36254_AT 36254forAAAGGAAGGAAACTCCTGACAGTCT 36254rev GCTTCAGAGATACAAATACAGTGTA (Seq. Id.37) ATACCA (Seq. Id. 38) 36672_at 36672for CTGGAGTAGAGTTCCTGGTTGCTT36672rev AAAGACACTTCAACCTCAAAACCAA (Seq. Id. 39) (Seq. Id. 40) 38694_at36694for CCTCAGTGGGTTCGTTAAAATCA 36694rev GGCATTTAATTGACCTCACAGAGA (Seq.Id. 41) (Seq. Id. 42) 36711_at 36711for GTGCCAATATGCCCTCCAAA 36711revGGCATCTCTTCAGTGCAATTTCT (Seq. Id. 43) (Seq. Id. 44) 37015_AT 37015forTTCTGAAATGTGACCCCCAAGT 37015rev GCTAAATGCAACTGTTCCTTTTCTA (Seq. Id. 45)TAA (Seq. Id. 46) 37183_at 37183for GGCAACATTTCCGAAGACCTT 37183revGCATACTCTAAAACGCACAGTGTGA (Seq. Id. 47) (Seq. Id. 48) 37230_AT 37230forCCTCCCTCCAGTGTCCACAT 37230rev AAGCAGGATTCTGGACATGGAA (Seq. Id. 49) (Seq.Id. 50) 38111_at 38111for AAGTGCTAATAATTAACTCAACCAGGT 38111revCTAGGTCTATATCCGTATGAAATGC CTA (Seq. Id. 51) ATT (Seq. Id. 52) 38112_g_at38112_gfor AAGTCACAATGAGTTTGGGCATATT 38112_grevACCATTTCCAGCCTAAACTACATAA (Seq. Id. 53) AA (Seq. Id. 54) 38404_at38404for CAGCTTTGACTTTATCTCCTGCTCTT 38404rev CCCTGGAGGCTGTGATCTCA (Seq.Id. 55) (Seq. Id. 56) 39114_at 39114for GAGTCTGAAGGACCCTAGTTCCTAGA39114rev TCTGTCCCTTCACCTCTGATCA (Seq. Id. 57) (Seq. Id. 58) 39397_at39397for AATTGTTTTTCGTCCGTTTGGTA 39397rev AATTGCCATATACGGCCAGTTAA (Seq.Id. 59) (Seq. Id. 60) 39579_at 39579for GCAGGAGCCTCACTGTGCAT 39579revACAGATGTGGCCCCGTTGTA (Seq. Id. 61) (Seq. Id. 62) 39599_at 39599p2FTGTACACATATGAACGCACAACATG 39599p2R GACCTCAGGAAAAGGCTTCAGA (Seq. Id. 63)(Seq. Id. 64) 39839_at 39839p2F GTCCAAACCAGCCGTCTGTT 39839p2RGCCTCTTTGCCATCTTGTGAA (Seq. Id. 65) (Seq. Id. 66) 40225_AT 40225forAAAGCCTCTGATTGTTGTTTCCTT 40225rev ACGCCCGAAACACCAAATAA (Seq. Id. 67)(Seq. Id. 68) 40382_at 40382for AGCAACAGGAAACTCATGGAGATT 40382revGAGCAGCGCTATGGTACAGAATACT (Seq. Id. 69) (Seq. Id. 70) 40425_at 40425forCAGCCTCAAAACGGGTCAGT 40425rev CTTCTTTCTCCGTCCCTCCG (Seq. Id. 71) (Seq.Id. 72) 40646_AT 40646for AATATCCCCTCCCAGTCACCTT 40646revAAAGCTGCTCCCATAGGCCT (Seq. Id. 73) (Seq. Id. 74) 41016_at 41016forTGACACCTGTGCATTAGATGCA 41016rev CATGGTGTGAGTCTGGGCCT (Seq. Id. 75) (Seq.Id. 76) 41439_at 41439for TGGTACAGGGTGCCTATTTTAGTCA 41439revTGTAGTTTCCAAAATGAACCCTTGT (Seq. Id. 77) (Seq. Id. 78) 41531_AT 41531forAACTGCCTTGTGTTCTGTGAGAAA 41531rev GCAGCATTGTAAGTTGTGATGCA (Seq. Id. 79)(Seq. Id. 80) 875_G_AT 875_Gfor TTGAACACTCACTCCACAACCC 875_GrevTTTCACATCAACAAACAAAATTCATT (Seq. Id. 81) ATAA (Seq. Id. 82)

RNA levels are then measured using Q-PCR. Briefly, cDNA is synthesizedusing random hexamers, diluted in a master mix containing TAQpolymerase, SybrGreen™ (Molecular Probes, Inc., Eugene, Oreg.),unlabeled nucleotides, buffer and water. The mixture is aliquotted intoTaqMan® plates (Perkin Elmer) and pairs of oligonucleotides are added tothe appropriate wells. Each sample is assayed in at least duplicatewells and every sample is assayed with every oligonucleotide pair wherethe transcriptase is omitted from the first reaction (noRT controls).The threshold cycle (CT) is calculated using Perkin Elmer software ABIPrism® 7700 Sequence Detection System Revison B. The CT value is definedas the cycle at which a statistically significant increase influorescence (from the SybrGreen™) is detected. A lower CT value isindicative of a higher mRNA concentration.

cDNA is separately prepared from all the N1 and S1 samples according toconventional methods. Yield is estimated using PicoGreen™ (MolecularProbes, Inc., Eugene, Oreg.) assays. Equal amounts of cDNA from all N1samples are mixed into one pooled sample, and equal amounts of cDNA fromall the S1 samples are mixed into a separate pooled sample. Q-PCR witholigonucleotides designed for GAPDH and the 38 genes identified from theGeneChip® experiments are executed in duplicate with no RT controls.Genes with a greater than 1 cycle difference in C_(T) between the twogroups are chosen for confirmation by Q-PCR on individual samples.

The 9 genes with the largest differences in CT between N1 and S1 pooledsamples are identified and Q-PCR is run on the individual cDNA samples.The individual CT values for these 9 genes plus the GAPDH (as control)are examined and t-test p-values are calculated to test the nullhypothesis that the two samples N1 and S1 are derived from the samepopulation. Five genes are found to be differentially expressed betweenthe normal and schizophrenic anterior cingulate samples. These geneshave the designation 34777_at, 36711_at, 39114_at, 39839_at and39579_at.

To confirm the differential resolution of these five genes, anindependent set of anterior cingulate samples is obtained (MarylandPsychiatric Research Clinic). Nine normal samples and 9 schizophrenicsamples are designated N2 and S2, respectively. For each of the 5 genes,plus GAPDH, standard curves are obtained by Q-PCR of a 2 fold dilutionseries of pooled normal cDNA with all 10 pairs of primers. Plotting CTagainst the logarithm of the starting amount of cDNA in microliters islinear. The equations of these standard curves can be used to calculatethe relative amounts of cDNA to a gene, GAPDH, that is regarded asunchanging between the samples. RNA samples are obtained from these newsamples and both the old and new samples are subjected to Q-PCR. Fromthe C_(T)'s the relative amounts of cDNA corrected for the amount ofGAPDH in each sample are calculated for all the genes and samples

The analysis of variance in the data demonstrated that three of thesegenes, 39114_at, 39839_at and 34777_at have consistently differentexpression levels in the normal and schizophrenic anterior cingulatesamples over both data sets with p-values of 0.001, 0.0013 and 0.0013,respectively.

BLAST (Basic Local Alignment Search Tool) searches of the publicsequence databases identified 39114_at, 34777_at, and 39839_at asdecidual protein induced by progesterone (DEPP), adrenomedullin, andcold shock domain protein A (csdA) respectively.

The foregoing description illustrates preferred embodiments of thepresent invention. It should be understood that those skilled in the artwill envision modifications of the embodiments that are covered by thefollowing claims.

1. A method of screening for schizophrenia in a population comprisingdetermining the magnitude of expression, in members of the population,of at least one gene selected from the group consisting of the geneencoding decidual protein induced by progesterone (DEPP), the geneencoding adrenomedullin and the gene encoding cold shock domain proteinA (csdA) in a sample and comparing the magnitude of expression to abaseline magnitude of expression of the gene, wherein increased geneexpression indicates the presence of schizophrenia.
 2. A method fordiagnosing schizophrenia in a host comprising determining the magnitudeof expression of at least one gene selected from the group consisting ofthe gene encoding decidual protein induced by progesterone (DEPP), thegene encoding adrenomedullin and the gene encoding cold shock domainprotein A (csdA) in a sample and comparing the magnitude of expressionto a baseline magnitude of expression of the gene, wherein increasedgene expression indicates the presence of schizophrenia.
 3. A methodaccording to claim 1 or 2 wherein the sample is taken from brain, spinalcord, lymphatic fluid, blood, urine or feces.
 4. A method according toclaim 3 wherein the sample is taken from the anterior cingulate.
 5. Amethod according to claim 1, 2, 3 or 4 wherein the sample is from ahuman.
 6. A method for treating schizophrenia comprising loweringexpression of at least one gene selected from the group consisting ofthe gene encoding decidual protein induced by progesterone (DEPP), thegene encoding adrenomedullin and the gene encoding cold shock domainprotein A (csdA) by administering to the host an expression loweringamount of antisense oligonucleotide or of siRNA or of ribozyme or ofnucleic acid molecules promoting triple helix formation with at leastone of said genes.
 7. Use of an antisense molecule or siRNA or aribozyme or a nucleic acid molecule promoting triple helix formationthat specifically inhibit the expression of DEPP, csdA or adrenomedullingenes for the manufacture of a medicament for the treatment ofschizophrenia.
 8. A method for treating schizophrenia comprisingreducing the amount of at least one protein selected from the groupconsisting of DEPP, adrenomedullin and csdA in a patient byadministering an effective amount of anti-DEPP, anti-adrenomedullinand/or anti-csdA antibody or functional antibody fragment sufficient tointerfere with the normal activity of the protein.
 9. Use of an antibodythat specifically binds an epitope of DEPP or csdA or adrenomedullinprior the manufacture of a medicament for the treatment of schizophrenia10. A method for treating schizophrenia according to claim 8 wherein theantibody or functional antibody fragment is selected from the groupconsisting of whole antibody, humanized antibody, chimeric antibody, Fabfragment, Fab′ fragment, F(ab′)₂ fragment, single chain Fv fragment anddiabody.
 11. A transgenic nonhuman animal expressing at least one of thegenes selected from the group consisting of the gene encoding decidualprotein induced by progesterone (DEPP), the gene encoding adrenomedullinand the gene encoding cold shock domain protein A (csdA) at higher thanbaseline levels and wherein said animal exhibits schizophrenic behavior.12. The animal of claim 11 wherein expression of said gene is enhancedby one or more alterations in regulatory sequences of the gene such thatthe gene is expressed at higher than baseline levels and wherein saidanimal exhibits schizophrenic behavior.
 13. The animal of claim 11wherein expression of said gene is enhanced by an increased copy numberof said gene, and wherein said animal exhibits schizophrenic behavior.14. A transgenic nonhuman animal according to claim 11, 12 or 13 whereinthe transgenic nonhuman animal is a mammal.
 15. A transgenic nonhumananimal according to claim 11, wherein the one or more alterationscomprises substitution of a promoter having a higher rate of expressionthan the native promoter of the gene.
 16. A transgenic nonhuman animalaccording to claim 15 wherein the promoter is an inducible promoter. 17.A transgenic nonhuman knockout animal whose genome comprises ahomozygous disruption in one or more genes selected from the groupconsisting of the gene encoding decidual protein induced by progesterone(DEPP), the gene encoding adrenomedullin and the gene encoding coldshock domain protein A (csdA), wherein said homozygous disruptionprevents the expression of the gene, and wherein said homozygousdisruption results in the transgenic knockout animal exhibitingdecreased expression levels of the one or more genes as compared to awild-type animal.
 18. A method of screening for a therapeutic agent thatmodulates symptoms of schizophrenia comprising administering a candidatecompound to a transgenic nonhuman animal according any of claim 1 to 16and determining the effect of the compound on symptoms associated withschizophrenia.
 19. A method of screening for a therapeutic agent thatmodulates symptoms of schizophrenia comprising combining a candidatecompound with a transgenic nonhuman animal according to claim 17 anddetermining the effect of the compound on symptoms associated withschizophrenia.
 20. A method of screening for a compound useful in thetreatment of schizophrenia comprising operatively linking a reportergene which expresses a detectable protein to a regulatory sequence for agene selected from the group consisting of DEPP, adrenomedullin and csdAto produce a reporter construct; transfecting a cell with the reporterconstruct; exposing the transfected cell to a test compound; andcomparing the level of expression of the reporter gene after exposure tothe test compound to the level of expression before exposure to the testcompound, wherein a lower level of expression after exposure isindicative of a compound useful for the treatment of schizophrenia.